PROJECT SUMMARY Obesity is a significant risk factor for the development of chronic disorders such as diabetes and cardiovascular disease. Current treatments are ineffectual given the worldwide increase in obesity rates to now epidemic proportions. The World Health Organization (WHO) reports that 1.9 billion (1 in 3) individuals globally are overweight and the rising cost to manage obesity-related diseases represented $147 billion dollars of the annual U.S. healthcare expenditure in 2008. The need for additional treatment options is critical. The goal of this proposal is to develop molecular treatments for obesity and associated metabolic disease. We take advantage of the thermogenic brown and beige fat biology that, when targeted, can increase energy expenditure and confer protection against obesity through the action of uncoupling protein 1 (UCP1). The mechanisms of this process, however, are not completely understood. Unveiling novel pathways and regulatory factors that can activate this process will maximize treatment options for individuals suffering from obesity and associated metabolic disease. Our previous work in murine models demonstrated that the transcription factor Yin Yang 1 (YY1) is critical for brown fat function. Adipose-specific ablation of YY1 (YY1bKO) results in significant thermogenic defects in the brown fat tissue, but the mechanism is unknown. In order to gain insight into the mechanism of action, we performed unbiased transcriptomic profiling in YY1bKO brown fat in search for novel thermogenic regulators and identified the gene nipsnap1 (4-nitrophenyl phosphatase domain and non-neuronal SNAP-25 like protein homolog1). Nipsnap1 is evolutionarily conserved across species, yet a clear functional annotation remains to be assigned. Our preliminary data show that Nipsnap1 exhibits a strong thermogenic profile and dramatically alters UCP1 protein levels. The goal of this proposal therefore is to identify the molecular function of Nipsnap1 in thermogenic adipose tissue in order to test the hypothesis that Nipsnap1 plays a significant role in thermogenesis. The studies proposed will use biochemical approaches in primary brown and beige adipocytes to detail the transcription and translation dynamics of Nipsnap1 in response to various thermogenic stimuli (Aim1). We will then continue with the functional characterization of Nipsnap1 through gain- and CRISPR-loss- of-function studies in addition to testing the coupled and uncoupled mitochondrial respiratory capacity of the system. Thermogenic adipose metabolomic profiling using high performance LC-MS/MS will also be assessed (Aim 2). Our studies will be the first to characterize the molecular function as well as the metabolomic-linked network of Nipsnap1 in thermogenic adipose tissue. The findings from this proposal will determine if Nipsnap1 can be leveraged towards a treatment for obesity and associated metabolic disorders.